Project Summary: The Na,K-ATPase alpha4 isoform has been found only in the membrane of male germ cells of rats and is most abundant in mature sperm. Specific pharmacological inhibition of this Na,K-ATPase isoform using the cardiac glycoside ouabain has been shown to completely inhibit the motility of rat sperm. The mechanism underlying the loss of motility of Na,K-ATPase alpha4 inhibited sperm is unclear, however indirect evidence suggests that an increase in intracellular [H+] is responsible. The Na,K-ATPase does not transport H+ itself therefore, the increase in intracellular [H+] associated with inhibition of the Na,K-ATPase alpha4 isoform is likely due to secondary inhibition of one of the Na+/H+ exchanger (NHE) isoforms found in sperm. The NHEs use the Na+ gradient established by the Na,K-ATPase to extrude H+ from the cell in exchange for extracellular Na+. Using rats and mice as models we w II asses the changes in intracellular ion concentrations in Na,K-ATPase inhibited sperm and examine the role that isoforms of the NHE and other Na,K-ATPase isoforms play in maintaining sperm motility. We will also characterize the Na,K-ATPase alpha4 isoform from humans with respect to its tissue distribution and its identity as a functional Na,K- ATPase alpha isoform. These studies will not only be important for defining the role that isoforms of the Na,K-ATPase and the NHE play in sperm motility but also may have medically relevant implications: If the human Na,K-ATPase alpha4 isoform plays the same role in human sperm as the rat isoform does in rat sperm it is likely that 1) a subset of infertile men are infertile due to inactivating mutations in the Na,K- ATPase alpha4 gene and 2) specific inhibitors of the Na,K-ATPase alpha4 isoform could be developed as male contraceptive agents. Relevance: Understanding the interactions between these proteins will further our understanding of the basic biology of sperm and will impact human reproductive biology. These studies could lead to understanding the underlying reasons for some forms of male infertility as well as to identifying male-specific contraceptives.